This invention relates to a novel method for transforming plant cells in vitro with Agrobacterium tumefaciens.
Certain strains of A. tumefaciens are responsible for crown gall disease in dicotyledons and gymnosperms. The disease is caused by a bacterium of such a strain attaching itself to the surface of a cell of a plant, at a wound site on the plant, and then transferring into the plant cell a large tumor-inducing (Ti) plasmid. The infected plant cell is thereby transformed into a gall (tumor) cell.
In crown gall disease, only portions of the Ti plasmid, transferred into a plant cell by an oncogenic strain of A. tumefaciens, are actually inserted into the plant cell's nuclear DNA. The inserted portions of the Ti plasmid (T-DNA), which can remain indefinitely in the plant cell's DNA, code for several functions which are expressed by the transformed plant cell. Such functions include tumorigenesis and the synthesis of abnormal opine metabolites that are specific to the Ti plasmid, transferred into the plant cell.
In recent years, the study of crown gall disease has become of importance because it may shed light on ways of controlling carcinogenesis and on ways of controlling gene expression in higher plants. In this regard, there has been particular interest in the possibility of using a Ti plasmid from an oncogenic strain of A. tumefaciens as a cloning vehicle or genetic vector for transforming higher plants. See Drummond, "Crown Gall Disease", Nature 281, 343-347 (1979).
However, only two ways have heretofore been known for transforming a plant cell with a Ti plasmid:
(1) By infecting an entire plant with crown gall disease (by applying oncogenic A. tumefaciens cells to a fresh wound on the plant) and then removing crown gall cells which develop on the plant; or PA1 (2) By isolating Ti plasmids and protoplasts of plant cells, transferring in vitro the Ti plasmids into the plant cell protoplasts, and then culturing the transformed protoplasts to produce plant cells. PA1 inoculating the plant cell with a strain of A. tumefaciens, containing the Ti plasmid, in the presence of an opine metabolite, that is normally synthesized only by a plant cell which has already been transformed by the Ti plasmid, or in the presence of the precursors of the opine metabolite; said opine metabolite or its precursors being capable of including conjugative activity of said Ti plasmid. PA1 (1) a plant cell, transformed with the specific Ti plasmid, normally synthesizes the opine metabolite; and PA1 (2) the opine metabolite or its precursors can induce conjugative activity of the specific Ti plasmid, i.e., cause the transfer of the Ti plasmid from one strain of A. tumefaciens to another by a process as described by Petit et al, "Substrate Induction of Conjugative Activity of Agrobacterium tumefaciens Ti Plasmids", Nature 271, 570-571 (1978).
Both ways have been relatively time-consuming and burdensome to carry out. As a result, it has been difficult to conduct experiments on plant cells with crown gall disease in the laboratory.
A more direct and simpler way of transforming a plant cell with a Ti plasmid has therefore been sought which would not require either the infection of an entire plant or the isolation of both the Ti plasmid and a protoplast of the plant cell.